Retroviral vectors for the transduction of autoregulated, bidirectional expression cassettes

Regulated transgene expression is increasingly used in research but is also needed for certain therapies. Regulatory systems are usually composed of t wo expression units, one bearing the gene of interest under control of a re gulatable promoter and the other, a constitutively expressed transactivator that modulates the activity of the regulatable promoter. Because the cotra nsfer of two independent elements is not efficient in primary cells, single transduction step vectors conferring regulatable gene expression cassettes would be helpful. We have developed retroviral vectors containing an autor egulatory bidirectional expression cassette that encodes all components nec essary for regulated expression of a gene of interest. The influence of the orientation of the reporter gene with respect to the viral long terminal r epeat (LTR) and the effect of transcriptionally inactive LTRs were investig ated using mouse leukemia virus (MLV) and self-inactivating (SIN)-based ret roviral vectors. Strict regulation was observed when the reporter was inser ted in antisense orientation with respect to the LTR, whereas a sense arran gement of the reporter resulted in a loss of regulation capacity. Expressio n and regulation of the antisense-orientated reporter gene were homogenous in infected cell pools and investigated cell clones. Long-term observations of infected cells over a period of 30 passages revealed stable expression and regulation. These autoregulated, bidirectional retroviral vectors combi ne the advantages of single-step transduction with strict regulation of the gene of interest in the infected target cells.